Method for detecting single nucleotide polymorphisms

ABSTRACT

An object of the present invention is to provide a method for rapidly and simply detecting single nucleotide polymorphisms. The present invention is a method for detecting single nucleotide polymorphisms, comprising analyzing wild-type and mutant-type products amplified by an AS-PCR method using ion-exchange chromatography.

TECHNICAL FIELD

The present invention relates to a method for rapidly and simplydetecting single nucleotide polymorphisms.

BACKGROUND ART

In recent years, techniques have been developed for analyzing singlenucleotide polymorphisms (SNP) which have been shown to be associatedwith various diseases and drug side effects; in the development thereof,it is an important factor to accurately detect single nucleotidepolymorphisms simply and in a short time.

An RFLP (Restriction Fragment Length Polymorphism) method is known as amethod for analyzing single nucleotide polymorphisms. The RFLP methodinvolves, when a restriction enzyme exists recognizing a gene mutationsite in a PCR (Polymerase Chain Reaction) amplification product,preparing primers in common sequence sites, performing amplification byholding polymorphisms in the PCR amplification product, cleaving theresultant PCR product with the restriction enzyme, and determining thepresence of polymorphisms based on the length of the fragments. However,the method has problems including that the use of restriction enzymeincreases analysis cost and prolongs time of the whole analysis. It alsohas problems including that the detection of the chain length differenceby electrophoresis complicates operation and prolongs time of the wholeanalysis.

In the fields of biochemistry, medicine, and the like, ion-exchangechromatography is used for the separation of biomacromolecules such asnucleic acids, proteins, and polysaccharides as a method capable ofaccurately detecting them simply and in a short time. The use ofion-exchange chromatography reduces complicated operation as requiredfor measurement by electrophoresis. Non Patent Literature 1 discloses amethod for separating nucleic acid-related compounds by high-performanceliquid chromatography. However, even the method disclosed in Non PatentLiterature 1 has a problem that it is difficult to sufficiently separatenucleic acids having chain lengths approaching to each other such assingle nucleotide polymorphisms.

CITATION LIST Non Patent Literature

Non Patent Literature 1: “Raifusaiensu Notameno Kosoku EkitaiKuromatogurafi Kiso To Jikken (High-Performance Liquid Chromatographyfor Life Science) (Basis and Experiment)”, Hirokawa Shoten, p. 323.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a method for rapidlyand simply detecting single nucleotide polymorphisms.

Solution to Problem

The present invention is a method for detecting single nucleotidepolymorphisms, comprising analyzing wild-type and mutant-type productsamplified by an AS-PCR method using ion-exchange chromatography.

The present invention will be described below in detail.

The present inventors have found that the wild-type and mutant-typeproducts amplified by an AS-PCR method can be analyzed usingion-exchange chromatography to detect single nucleotide polymorphismsrapidly and simply, thereby accomplishing the present invention.

The AS-PCR (Allele Specific-PCR) method is a method for detecting genepolymorphism (particularly, single nucleotide polymorphisms) using asequence-specific amplification reaction. Specifically, PCR is performedin such a manner that a nucleotide sequence of a single nucleotidepolymorphism desired to be detected is located at the 3′ end of primer.When the sequence of the target nucleic acid is completely complementaryto the primer, an extension reaction by DNA polymerase occurs. Incontrast, when the sequence of the target nucleic acid is incompletelycomplementary to the primer, the extension reaction of DNA polymerase isinhibited. Thus, it is a method which involves using two primers, whichhave a wild-type or mutant-type nucleotide sequence of a singlenucleotide polymorphism at the 3′ end, to perform the determination ofthe single nucleotide polymorphism based on the results of theamplification reaction. The AS-PCR method can use a method as disclosedin “Nature, 324, p. 163-166, 1986”.

The method for detecting single nucleotide polymorphisms according tothe present invention uses ion-exchange chromatography.

The eluent used for ion-exchange chromatography preferably contains aguanidine salt derived from guanidine represented by formula (1) below.

Examples of the guanidine salt include guanidine hydrochloride,guanidine sulfate, guanidine nitrate, guanidine carbonate, guanidinephosphate, guanidine thiocyanate, guanidine sulfamate, aminoguanidinehydrochloride, and aminoguanidine bicarbonate. Guanidine hydrochlorideand guanidine sulfate are preferably used, among these.

The concentration of a guanidine salt in the eluent when analyzed may beproperly adjusted in accordance with a substance to be detected;however, it is preferably 2,000 mmol/L or less.

Specifically, a method can be mentioned which involves performinggradient elution in the guanidine salt concentration range of 0 to 2,000mmol/L. Thus, it is not necessary that the concentration of theguanidine salt in starting analysis is 0 mmol/L, and it is also notnecessary that the concentration of the guanidine salt in terminatinganalysis is 2,000 mmol/L.

The method of gradient elution may be a low-pressure gradient method ora high-pressure gradient method; however, a method is preferable whichinvolves carrying out elution while performing precise concentrationadjustment by the high-pressure gradient method.

The guanidine salt may be added alone to the eluent or in combinationwith another salt. Examples of the salt capable of being used incombination with the guanidine salt include salts consisting of halidesand alkali metals, such as sodium chloride, potassium chloride, sodiumbromide, and potassium bromide, salts consisting of halides and alkaliearth metals, such as calcium chloride, calcium bromide, magnesiumchloride, and magnesium bromide, and inorganic acid salts such as sodiumperchlorate, potassium perchlorate, sodium sulfate, potassium sulfate,ammonium sulfate, sodium nitrate, and potassium nitrate. Organic saltssuch as sodium acetate, potassium acetate, sodium succinate, andpotassium succinate may also be used.

A known buffer or an organic solvent can be used as a buffer used in aneluent; specific examples thereof include Tris-hydrochloric acid buffer,TE buffer consisting of Tris and EDTA, TAE buffer consisting of Tris,acetic acid, and EDTA, and TBA buffer consisting of Tris, boric acid,and EDTA.

The pH of the eluent is not particularly limited, as long as it is in arange that allows the separation of nucleic acid chains by anionicexchange.

The filler used for ion-exchange chromatography is preferably one havingcationic groups introduced into at least the surface of base materialparticles, and more preferably one having strong cationic groups andweak anionic groups on at least the surface of base material particles.

As used herein, the “strong cationic group” means a cationic groupdissociating in the wide pH range of 1 to 14. Thus, the strong cationicgroup can retain a dissociated (cationized) state without being affectedby the pH of the aqueous solution.

Examples of the strong cationic group include quaternary ammoniumgroups. Specific examples thereof include trialkylammonium groups suchas a trimethylammonium group, a triethylammonium group, and adimethylethylammonium group.

Examples of counter ions for the strong cationic group include halideions such as chloride ion, bromide ion, and iodide ion.

The amount of the strong cationic group is not particularly limited;however, the lower limit thereof per dry weight of the filler ispreferably 1 μeq/g and the upper limit is preferably 500 μeq/g. A strongcationic group amount of less than 1 μeq/g may weaken the retainingforce of the filler and deteriorate separation performance. A strongcationic group amount of more than 500 μeq/g may pose problems of makingthe retaining force of the filler too strong, thereby not easily causingthe elution of a substance, prolonging analysis time, and the like.

As used herein, the “weak anionic group” means an anionic group having apKa of 3 or more. Thus, the weak anionic group described above isaffected by the pH of the aqueous solution, by which the dissociatedstate thereof changes. A pH of more than 3 causes the dissociation ofthe proton of the carboxy group and increases the percentage thereofhaving a minus charge. Conversely, a pH of less than 3 increases thepercentage of the carboxy group in an undissociated state in which theproton of the carboxy group is bonded.

Examples of the weak anionic group described above include a carboxygroup and a phosphoric acid group. A carboxy group is preferable, amongthese.

Examples of methods for introducing carboxy groups into at least thesurface of base material particles, which can be used, include knownmethods such as a method involving copolymerizing a monomer having acarboxy group, a method involving hydrolyzing the ester moiety of amonomer, a method involving forming a carboxy group by ozonated watertreatment, a method involving forming a carboxy group using ozone gas, amethod involving forming a carboxy group by plasma treatment, a methodinvolving reacting a silane coupling agent having a carboxy group, and amethod involving copolymerizing a monomer having an epoxy group andforming a carboxy group by ring-opening of the epoxy group. Among these,a method involving forming a carboxy group by ozonated water treatmentis preferably used when the base material particle has hydrophobicstructural portions, particularly carbon-carbon double bonds.

The method involving forming a carboxy group by ozonated water treatmentwill be described.

Ozone has high reactivity with a double bond, and the ozone reactingwith the double bond forms ozonide as an intermediate, followed by theformation of a carboxy group and the like.

Ozonated water means what is formed by dissolving ozone gas in water.

Ozonated water can be used to simply oxidize the particle surface bymerely dispersing the particles in the ozonated water. As a result,hydrophobic structural portions in the base material particle can beconsidered to be oxidized to form hydrophilic groups such as a carboxygroup, a hydroxyl group, an aldehyde group, and a keto group.

Ozone has a strong oxidation effect; treatment with ozonated water ispreferable because it can more uniformly oxidize the particle surfaceand causes the more uniform formation of carboxy groups than treatmentwith ozone gas.

The concentration of dissolved ozone in the ozonated water is notparticularly limited; however, the lower limit thereof is preferably 20ppm. A dissolved ozone concentration of less than 20 ppm requires a longtime to form a carboxy group, or cannot sufficiently suppress thenon-specific adsorption or the like of a substance to be detected sinceit causes the insufficient formation of a carboxy group. The lower limitof the dissolved ozone concentration is more preferably 50 ppm.

The ozonated water can be prepared, for example by a method involvingcontacting raw material water with ozone gas via an ozone gas-permeablemembrane allowing only gas to pass therethrough and blocking thepermeation of liquid as described, for example, in JP 2001-330969 A.

Under alkali conditions, it can be considered that the carboxy groupsintroduced into the surface of the base material particle are in anearly dissociated state and produce weak cation exchange interactionwith a few cations in a nucleic acid base.

It can also be considered that treatment with ozonated water causes theformation of hydrophilic groups such as a hydroxyl group, an aldehydegroup, and a keto group in addition to a carboxy group and the presenceof these hydrophilic groups weakens hydrophobic interaction actingbetween the filler surface and the nucleic acid.

Thus, it can be considered that the use of a filler having strongcationic groups and weak anionic groups on at least the surface improvesseparation performance by the action of the weak cation exchangeinteraction and the weakening of the hydrophobic interaction asdescribed above in addition to the anion exchange interaction actingbetween the filler surface and a nucleic acid as the main interaction.

The amount of the weak anionic groups introduced into at least thesurface of the base material particle is not particularly limitedprovided that it is smaller than or equal to the amount of the strongcationic group.

The base material particle which can be used is, for example, asynthetic polymer fine particle obtained using a polymerizable monomeror the like, and an inorganic fine particle such as silica; however, itis preferably one consisting of a hydrophobic cross-linked polymerparticle consisting of an organic synthetic polymer and a layerconsisting of a hydrophilic polymer having ion exchange groupscopolymerized on the surface of the hydrophobic cross-linked polymerparticle.

The hydrophobic cross-linked polymer may be a hydrophobic cross-linkedpolymer obtained by homopolymerizing one hydrophobic cross-linkablemonomer, a hydrophobic cross-linked polymer obtained by copolymerizingtwo or more hydrophobic cross-linkable monomers, or a hydrophobiccross-linked polymer obtained by copolymerizing at least one hydrophobiccross-linkable monomer and at least one hydrophobic non-cross-linkablemonomer.

The hydrophobic cross-linkable monomer is not particularly limitedprovided that it has 2 or more vinyl groups in one molecule of themonomer. Examples thereof include di(meth)acrylic esters such asethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate,propylene glycol di(meth)acrylate, and polypropylene glycoldi(meth)acrylate; tri(meth)acrylic esters or tetra(meth)acrylic esterssuch as tetramethylol methane tri(meth)acrylate, trimethylol propanetri(meth)acrylate, and tetramethylol methane tetra(meth)acrylate; andaromatic compounds such as divinylbenzene, divinyltoluene,divinylxylene, and divinylnaphthalene.

As used herein, the “(meth)acrylic” means “acrylic or methacrylic”, andthe “(meth)acrylate ” means “acrylate or methacrylate”.

The hydrophobic non-cross-linkable monomer is not particularly limitedprovided that it is a non-cross-linkable polymerizable organic monomerhaving hydrophobic properties; examples thereof include (meth)acrylicesters such as methyl(meth)acrylate, ethyl(meth)acrylate,propyl(meth)acrylate, isopropyl(meth)acrylate, butyl(meth)acrylate, andt-butyl(meth)acrylate, and styrene monomers such as styrene andmethylstyrene.

When the hydrophobic cross-linked polymer consists of a copolymer of ahydrophobic cross-linkable monomer and a hydrophobic non-cross-linkablemonomer, the lower limit of the content of the segment derived from thehydrophobic cross-linkable monomer in the hydrophobic cross-linkedpolymer is preferably 10% by weight, more preferably 20% by weight.

The hydrophilic polymer having ion exchange groups is composed of ahydrophilic monomer having an ion exchange group and shall contain thesegment derived from a hydrophilic monomer having one or more kinds ofion exchange groups. Thus, Methods for producing a hydrophilic polymerhaving ion exchange groups include a method involving homopolymerizing ahydrophilic monomer having an ion exchange group and a method involvingcopolymerizing a hydrophilic monomer having an ion exchange group and ahydrophilic monomer not having an ion exchange group.

The hydrophilic monomer having an ion exchange group is preferably onehaving a strong cationic group and more preferably one having aquaternary ammonium group. Specific examples thereof include ethylmethacrylate trimethylammonium chloride, ethyl methacrylatetriethylammonium chloride, ethyl methacrylate dimethylethylammoniumchloride, ethyl acrylate trimethylammonium chloride, ethyl acrylatetriethylammonium chloride, ethyl acrylate dimethylethylammoniumchloride, acrylamide ethyltrimethylammonium chloride, acrylamideethyltriethylammonium chloride, and acrylamideethyldimethylethylammonium chloride.

The average particle diameter of the filler is not particularly limited;however, the preferable lower limit thereof is 0.1 μm, and thepreferable upper limit is 20 μm. An average particle diameter of thefiller of less than 0.1 μm increases the internal pressure of the columnand may cause poor separation. An average particle diameter of thefiller of more than 20 μm makes dead volume in the column too large andmay cause poor separation.

As used herein, the average particle diameter refers to the volumeaverage particle diameter, and can be measured using a particle sizedistribution analyzer (AccuSizer780 from Particle Sizing Systems).

According to the detection method for single nucleotide polymorphisms ofthe present invention, the size of the product amplified by the AS-PCRmethod is preferably 200 bp or less. A size of the product amplified bythe AS-PCR method of more than 200 bp may prolong amplification time ofPCR and analysis time in ion-exchange chromatography or may causeinsufficient separation performance. The size of the product amplifiedby the AS-PCR method is preferably 100 bp or less.

According to the detection method for single nucleotide polymorphisms ofthe present invention, the size difference of the products (differencein chain length) between the wild-type and mutant-type amplified by theAS-PCR method is preferably 10 bp or less. When AS primers are designedso that the size difference of the products between the amplifiedwild-type and mutant-type exceeds 10 bp, desired amplification productsmay not be obtained due to a non-specific amplification reaction and thelike.

Advantageous Effects of Invention

According to the present invention, a method can be provided for rapidlyand simply detecting single nucleotide polymorphisms.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a pair of chromatograms obtained by separating and detectingwild-type 76 bp and mutant-type 79 bp in UGT1A1*6 region using anionexchange column 1 in Example 1.

FIG. 2 is a pair of chromatograms obtained by separating and detectingwild-type 76 bp and mutant-type 79 bp in UGT1A1*6 region using anionexchange column 2 in Example 1.

FIG. 3 is a pair of chromatograms obtained by separating and detectingwild-type 271 bp and mutant-type 274 bp in UGT1A1*6 region using anionexchange column 1 in Reference Example 1.

FIG. 4 is a pair of chromatograms obtained by separating and detectingwild-type 271 bp and mutant-type 274 bp in UGT1A1*6 region using anionexchange column 2 in Reference Example 1.

FIG. 5 is a pair of chromatograms obtained by separating and detectingwild-type 76 bp and mutant-type 79 bp in UGT1A1*6 region using anionexchange column 1 in Reference Example 2.

DESCRIPTION OF EMBODIMENTS

The present invention will be described below in further detail withreference to Examples. However, the present invention is not limited toonly these Examples.

(Provision of Anion Exchange Column) (Anion Exchange Column 1)

In a reactor provided with a stirrer, 300 g of tetraethylene glycoldimethacrylate (from Shin-Nakamura Chemical Co., Ltd.), 100 g oftriethylene glycol dimethacrylate (from Shin-Nakamura Chemical Co.,Ltd.), and 1.0 g of benzoyl peroxide (from Kishida Chemical Co., Ltd.)were added to 2,000 mL of aqueous solution of 3% by weight polyvinylalcohol (from Nippon Synthetic Chemical Industry Co., Ltd.). The mixturewas heated while stirring and polymerized at 80° C. for 1 hour in anatmosphere of nitrogen. Then, 100 g of ethyl methacrylatetrimethylammonium chloride (from Wako Pure Chemical Industries Ltd.) asa monomer having a strong cationic ion exchange group (a quaternaryammonium group) was dissolved in ion-exchange water, and the resultantsolution was further added into the reactor. Subsequently, the solutionwas polymerized at 80° C. for 2 hours in an atmosphere of nitrogen whilestirring to provide a polymer composition. The resultant polymercomposition was washed with water and acetone to provide hydrophiliccoated polymer particles having quaternary ammonium groups on thesurface of base material particles.

10 g of the resultant coated polymer particles were immersed in 300 mLof ozonated water having a dissolved ozone concentration of 100 ppm andstirred for 30 minutes. After stirring, centrifugation was performedusing a centrifuge (“Himac CR20G” from Hitachi, Ltd.), and thesupernatant was removed. This operation was repeated 2 times, andozonated water treatment was applied to the coated polymer particles toprovide a filler for ion-exchange chromatography in which quaternaryammonium groups and carboxy groups coexist.

The ozonated water was prepared using an ozonated water productionsystem in which 400 hollow tube-shaped ozone gas permeable membranes 0.5mm in inside diameter, 0.04 mm in thickness, and 350 cm in length wereenclosed in a cylindrical mantle 15 cm in inside diameter and 20 cm inlength (from Sekisui Chemical Co., Ltd.).

When the resultant filler for ion-exchange chromatography was measuredusing a particle size distribution analyzer (“Accusizer780” fromParticle Sizing Systems), the average particle diameter thereof wasfound to be 10 μm.

The following column (anion exchange column 1) was provided using theresultant filler for ion-exchange chromatography.

Column size: 4.6 mm in inside diameter×20 mm

Ion exchange group: quaternary ammonium group

(Anion Exchange Column 2)

The following column as a commercially available column was provided.

Product name: TSK-gel DNA-STAT (from Tosoh Corporation)

Column size: 4.6 mm in inside diameter×100 mm in length

Ion exchange group: quaternary ammonium group

EXAMPLE 1

The separation and detection of wild-type 76 bp and mutant-type 79 bp inUGT1A1*6 region was performed in Example 1.

(AS-PCR Amplification)

Wild-type and mutant-type amplification products were obtained usingAS-PCR conditions as described below.

(1) Reagent

-   AccuPrime Taq DNA Polymerase High Fidelity (from Invitorgen, Lot.    760816)-   10×AccuPrime PCR Buffer I-   AccuPrime Taq DNA Polymerase High Fidelity (5 U/μL) UGT1A1*6 primer    (from Operon Biotechnologies)

Forward (wild-type) (10 pmol/μL): (SEQ ID NO: 1)5′-(cgcctcgttgtacatcagagcgg)-3′ Forward (mutant-type) (10 pmol/μL):(SEQ ID NO: 2) 5′-(ctgacgcctcgttgtacatcagagcga)-3′ Reverse (10 pmol/μL):(SEQ ID NO: 3) 5′-(cacatcctccctttggaatggca)-3″

-   Nuclease-free Water (not DEPC-treated) (from Ambion, Lot. 0803015)-   UGT1A1 gene wild-type sequence-inserted plasmid (1×10⁶ copies/μL)-   UGT1A1 gene mutant-type sequence-inserted plasmid (1×10⁶ copies/μL)

(2) Preparation

One (1) μL of each UGT1A1 gene sequence-inserted plasmid was added to asolution prepared by adding Nuclease-free Water to 5 μL of 10×AccuPrimePCR Buffer I, 1 μL of the Forward primer, and 1 μL of the Reverse primerto make a total volume of 49 μL of a reaction solution.

(3) Reaction

PCR reaction was performed using C1000 (from BIO-RAD Laboratories). Thetemperature cycle is as described below.

The template was heat-degenerated at 94° C. for 30 seconds; theamplification cycle of 94° C. for 15 seconds, 62° C. for 15 seconds, and68° C. for 30 seconds was repeated 40 times; and finally, incubation wascarried out at 68° C. for 5 minutes. The samples were stored at 4° C.until use.

After AS-PCR amplification, bands derived from the amplification productwere identified at about 80 bp by electrophoresis (“Mupid-ex” fromAdvance Co., Ltd.). The amplification product size was determined using20 bp DNA Ladder Marker (from Takara Bio Inc.).

(HPLC Analysis)

Using the provided anion exchange column, the AS-PCR amplificationproducts were separated and detected under the following conditions.

-   System: LC-20A series (from Shimadzu Corporation)-   Eluent: eluent A 25 mmol/L Tris-Hydrochloride buffer (pH 7.5)

eluent B 25 mmol/L Tris-Hydrochloride buffer (pH 7.5)+1 mol/L guanidinehydrochloride

-   Analysis time: Analysis time was 10 minutes when anion exchange    column 1 was used.    -   Analysis time was 20 minutes when anion exchange column 2 was        used.-   Elution method: the mixing ratio of eluent B was linearly increased    using the following gradient conditions.    -   Conditions when anion exchange column 1 was used    -   0 minute (eluent B 40%)→10 minutes (eluent B 50%)    -   Conditions when anion exchange column 2 was used    -   0 minute (eluent B 70%)→20 minutes (eluent B 90%)-   Analyte: Wild-type 76 bp in UGT1A1*6 region

Mutant-type 79 bp in UGT1A1*6 region

-   Flow rate: 0.5 mL/min. (when anion exchange column 1 was used)

1.0 mL/min. (when anion exchange column 2 was used)

-   Detection wavelength: 260 nm-   Sample injection volume: 10 μL

REFERENCE EXAMPLE 1

The separation and detection of wild-type 271 bp and mutant-type 274 bpin UGT1A1*6 region were performed in Reference Example 1.

(AS-PCR Amplification)

Wild-type and mutant-type amplification products were obtained using thefollowing AS-PCR conditions.

(1) Reagent

AccuPrime Taq DNA Polymerase High Fidelity (from Invitrogen, Lot.760816)

-   10×AccuPrime PCR Buffer I-   AccuPrime Taq DNA Polymerase High Fidelity (5 U/μL) UGT1A1*6 primer    (from Operon Biotechnologies)

Forward (wild-type) (10 pmol/μL): (SEQ ID NO: 1)5′-(cgcctcgttgtacatcagagcgg)-3′ Forward (mutant-type) (10 pmol/μL):(SEQ ID NO: 2) 5′(ctgacgcctcgttgtacatcagagcga)-3′ Reverse (10 pmol/μL):(SEQ ID NO: 4) 5′-(gaaagggtccgtcagcatgac)-3″

-   Nuclease-free Water (not DEPC-treated) (from Ambion, Lot. 0803015)-   UGT1A1 gene wild-type sequence-inserted plasmid (1×10⁶ copies/μL)-   UGT1A1 gene mutant-type sequence-inserted plasmid (1×10⁶ copies/μL)

(2) Preparation

One (1) μL of each UGT1A1 gene sequence-inserted plasmid was added to asolution prepared by adding Nuclease-free Water to 5 μL of 10×AccuPrimePCR Buffer I, 1 μL of the Forward primer, and 1 μL of the Reverse primerto make a total volume of 49 μL of a reaction solution.

(3) Reaction

PCR reaction was performed using C1000 (from BIO-RAD Laboratories). Thetemperature cycle is as described below.

The template was heat-degenerated at 94° C. for 30 seconds; theamplification cycle of 94° C. for 15 seconds, 62° C. for 15 seconds, and68° C. for 30 seconds was repeated 40 times; and finally, incubation wascarried out at 68° C. for 5 minutes. The samples were stored at 4° C.until use.

After AS-PCR amplification, bands derived from the amplification productwere identified at about 270 bp (between 200 bp and 300 bp) byelectrophoresis (“Mupid-ex” from Advance Co., Ltd.). The amplificationproduct size was determined using 20 bp DNA Ladder Marker (from TakaraBio Inc.).

(HPLC Analysis)

Using the provided anion exchange column, the AS-PCR amplificationproducts were separated and detected under the following conditions.

-   System: LC-20A series (from Shimadzu Corporation)-   Eluent: eluent A 25 mmol/L Tris-Hydrochloride buffer (pH 7.5)

eluent B 25 mmol/L Tris-Hydrochloride buffer (pH 7.5)+1 mol/L guanidinehydrochloride

-   Analysis time: Analysis time was 10 minutes when anion exchange    column 1 was used.    -   Analysis time was 20 minutes when anion exchange column 2 was        used.-   Elution method: the mixing ratio of eluent B was linearly increased    using the following gradient conditions.    -   Conditions when anion exchange column 1 was used    -   0 minute (eluent B 60%)→10 minutes (eluent B 80%)    -   Conditions when anion exchange column 2 was used    -   0 minute (eluent B 80%)→20 minutes (eluent B 100%)-   Analyte: Wild-type 271 bp in UGT1A1*6 region

Mutant-type 274 bp in UGT1A1*6 region

-   Flow rate: 0.5 mL/min. (when anion exchange column 1 was used)

1.0 mL/min. (when anion exchange column 2 was used)

-   Detection wavelength: 260 nm-   Sample injection volume: 10 μL

COMPARATIVE EXAMPLE 1

The separation and detection of wild-type 76 bp and mutant-type 96 bp inUGT1A1*6 region were attempted to be performed in Comparative Example 1.

(1) Reagent

AccuPrime Tag DNA Polymerase High Fidelity (from Invitorgen, Lot.760816)

-   10×AccuPrime PCR Buffer I-   AccuPrime Tag DNA Polymerase High Fidelity (5 U/μL)-   UGT1A1*6 primer (from Operon Biotechnologies)

Forward (wild-type) (10 pmol/μL): (SEQ ID NO: 1)5′-(cgcctcgttgtacatcagagcgg)-3′ Forward (mutant-type) (10 pmol/μL):(SEQ ID NO: 5) 5′- (atagttgtcctagcacctgacgcctcgttgtacatcagagcga)-3″Reverse (10 pmol/μL): (SEQ ID NO: 3) 5′-(cacatcctccctttggaatggca)-3′

-   Nuclease-free Water (not DEPC-treated) (from Ambion, Lot. 0803015)-   UGT1A1 gene wild-type sequence-inserted plasmid (1×10⁶ copies/μL)-   UGT1A1 gene mutant-type sequence-inserted plasmid (1×10⁶ copies/μL)

(2) Preparation

One (1) μL of each UGT1A1 gene sequence-inserted plasmid was added to asolution prepared by adding Nuclease-free Water to 5 μL of 10×AccuPrimePCR Buffer I, 1 μL of the Forward primer, and 1 μL of the Reverse primerto make a total volume of 49 μL of a reaction solution.

(3) Reaction

PCR reaction was performed using C1000 (from BIO-RAD Laboratories). Thetemperature cycle is as described below.

The template was heat-degenerated at 94° C. for 30 seconds; theamplification cycle of 94° C. for 15 seconds, 62° C. for 15 seconds, and68° C. for 30 seconds was repeated 40 times; and finally, incubation wascarried out at 68° C. for 5 minutes. The samples were stored at 4° C.until use.

When, after AS-PCR amplification, the amplification product wasdetermined by electrophoresis (“Mupid-ex” from Advance Co., Ltd.), manybands likely to indicate non-specific amplification were identified.This means that the AS-PCR amplification was not properly performed.Thus, HPLC analysis was not carried out.

REFERENCE EXAMPLE 2

The separation and detection of wild-type 76 bp and mutant-type 79 bp inUGT1A1*6 region were performed in Reference Example 2.

HPLC analysis was performed using anion exchange column 2 in the sameway as Example 1, except that the salt added to eluent B was sodiumchloride in place of guanidine hydrochloride.

The chromatograms obtained by separating and detecting wild-type 76 bpand mutant-type 79 bp in UGT1A1*6 region in Example 1 are shown in FIG.1 (when anion exchange column 1 is used) and FIG. 2 (when anion exchangecolumn 2 is used). The results of FIGS. 1 and 2 show that both columnscould favorably separate and detect the wild-type 76 bp and mutant-type79 bp in UGT1A1*6 region amplified by AS-PCR. Particularly, the use ofanion exchange column 1 could almost completely separate and detect themin a short time.

The chromatograms obtained by separating and detecting wild-type 271 bpand mutant-type 274 bp in UGT1A1*6 region in Reference Example 1 areshown in FIG. 3 (when anion exchange column 1 is used) and FIG. 4 (whenanion exchange column 2 is used). The results of FIGS. 3 and 4 show thatthe wild-type 271 bp and mutant-type 274 bp in UGT1A1*6 region amplifiedby AS-PCR could not be separated in contrast to Example 1. It can beconsidered that the reason for this lies in the fact that, compared tothe size of the AS-PCR amplification products, the difference in thechain length between the wild-type and the mutant-type was relativelysmall.

The chromatograms obtained by separating and detecting wild-type 76 bpand mutant-type 79 bp in UGT1A1*6 region in Reference Example 2 areshown in FIG. 5. When sodium chloride was added in place of guanidinehydrochloride to the eluent B, the wild-type 76 bp and the mutant-type79 bp could not be separated.

INDUSTRIAL APPLICABILITY

According to the present invention, a method can be provided for rapidlyand simply detecting single nucleotide polymorphisms.

1. A method for detecting single nucleotide polymorphisms, comprising analyzing wild-type and mutant-type products amplified by an AS-PCR method using ion-exchange chromatography.
 2. The method for detecting single nucleotide polymorphisms according to claim 1, wherein the size of the products amplified by an AS-PCR method is 200 bp or less and the difference in the chain length between the wild-type and the mutant-type is 10 bp or less.
 3. The method for detecting single nucleotide polymorphisms according to claim 1, comprising using an eluent containing a guanidine salt derived from guanidine represented by formula (1) below in ion-exchange chromatography:


4. The method for detecting single nucleotide polymorphisms according to claim 3, wherein the guanidine salt is guanidine hydrochloride or guanidine sulfate.
 5. The method for detecting single nucleotide polymorphisms according to claim 2, comprising using an eluent containing a guanidine salt derived from guanidine represented by formula (1) below in ion-exchange chromatography:


6. The method for detecting single nucleotide polymorphisms according to claim 5, wherein the guanidine salt is guanidine hydrochloride or guanidine sulfate. 